Protozoans which invade the blood stream and tissues of higher animals and live parasitically therein may cause serious diseases in the host. In fact, many of the diseases that have plagued mankind for centuries are due to protozoan parasites. African sleeping sickness, for example, is generally caused by Trypanosoma brucei gambiense which is spread by the bite of the tsetse fly, a blood-sucking insect. Another species of this genus, Trypanosoma brucei brucei infects domestic animals; death follows infection for horses, cattle and pigs. Kala-azar is a serious disease caused by Leishmania donovani which is spread by sand-flies. Chagas disease is wide-spread in tropical areas where T. cruzi infections occur.
A sensitive, rapid assay is needed for diagnosis of these diseases. Current techniques involve the isolation and cultivation of individual parasites which is time-consuming and often un-reliable (Chance, M. L., et al. Ann. Trop. Med. Parasitol. 68: 307 (1974); Martin, E., et al., Protozoology 23: 600 (1978); Miles, M. A., et al., Trans. R. Soc. Trop. Med. Hyg. 74, 243 (1979); Lainson, R., Trans. R. Soc. Trop. Med. Hyg., 75: 530 (1981)). Lesions due to infection by Leishmania species may be analyzed for parasites, by direct examination but this method does not allow identification of species causing more serious forms of the disease. Moreover other species, such as Tripanosomae do not express themselves in lesions and so require sensitive serological tests. The more reliable of those are difficult to use in large scale (immunofluorescence), or display cross-reactivity with other microorganisms (complement fixation, agglutination).
In addition, a major health problem has been created in areas of high infection when blood is collected for transfusion purposes. Since blood is a carrier of the parasites, blood from an infected individual may be unknowingly transferred to a healthy individual. Chagas disease, particularly, has been found to be a major problem for blood banks in countries where the disease is endemic, with many documented cases of transmission of the infection by blood transfusions. Current assays for these protozoan parasites have neither specificity nor sensitivity to detect parasites in blood and are thus not useful for screening of blood to be used for transfusion.
The economic toll to agriculture due to these protozoan parasites of domestic animals is immense. Infection by Trypanosoma brucei, for example, is fatal to cattle unless treated early in the course of disease. There is no currently available sensitive diagnostic assay for infection, so many animals are lost. Moreover, the effectiveness of chemotherapy in both humans and animals cannot be currently monitored so there is often needless reccurrence of the disease.
Wirth and Pratt (Proc. Nat'l. Acad. Sci. USA 79 6999 (1983) have described a hybridization assay for the detection of Leishmania parasites using probes to parasite kinetoplast DNA. This assay detects parasites in cutaneous lesions at a sensitivity level of 1,000-10,000 parasites per biopsy specimen. The specimens are collected by touch-blotting of nitrocellulose sheets over a small area of infected skin. However, this method is not sensitive enough to detect small numbers of parasites and relies on probes that have to be purified from the parasites themselves. This requires growth of these organisms in large quantities in the laboratory. Moreover, the method cannot be extended to parasites other than Trypanosomatidae, since these are the only ones to possess a kinetoplast.
A more sensitive assay has been sought for both Leishmania and other protozoan parasites which will be sensitive, specific, and thus be useful in early diagnosis of infection, will identify species of parasites more likely to induce severe disease, and aid evaluation of chemotherapy and screening of blood bank samples.
The complementary nature of the double-stranded DNA which comprises the genome plays a fundamental role in the duplication of the cell and in the transcription and translation of genetic information. In the laboratory, complementary strands of DNA may be readily dissociated, and under appropriate conditions of cation concentration, temperature, DNA concentration and fragment size, may be reproducibly re-associated. This complementary nature of DNA is the basis of a sensitive assay for genetic material. The Southern blot method, for instance, utilizes hybridization probes which are complementary DNA strands. A related assay, the Northern blot method, utilizes the ability of DNA to associate with complementary RNA probes. Detection devices employing RNA or DNA tagged with either a radioactive element or biotin have been developed (Gardner, L., Biotechniques March Volume 38 (1983); Langer, P. R., et al., Proc. Nat'l. Acad. Sci. U.S.A., 78 6633 (1981).
The reproducibility and reliability of the blot methods in general is due to the accuracy with which complementary strands recognize one another. However, each genome contains a large number of nucleotide pairs. The calf genome, for example, contains 3.2.times.10.sup.19 nucleotide pairs (McCarthy, B. J., Progr. Nucleic Acid Res. Mol. Biol. 4, 129 (1965). It might seem that the probability of one chain finding and combining with its complement would be small. Surprisingly, however, it has been found that in genomes of some organisms the DNA occurs in repeated sequences that may account for a large proportion of the total genome (Britten, R. J. et al., Carnegie Inst. Wash. Year Book, 66, 73 (1967); ibid 65, 73 (1966)). Ten percent of the mouse genome, for example, consists of a million copies of a short nucleotide sequence (Waring, M. Et. al., Science, 154, 791 (1966)). It has been observed that many other species also contain repetitive DNA elements (Britten et al., Science 161, 529 (1968)).
Since the blot hybridization methods detect DNA sequences, it follows that sensitivity and specificity of assays are enhanced if a probe which recognizes a repetitive sequence can be obtained. Moreover, if this repetitive sequence is species specific, the probe recognizing it is also specific. Protozoan parasites containing such species-specific repetitive nuclear DNA elements have been sought.